*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Mycobacterium tuberculosis is the leading bacteria that causes the pathogenic disease tuberculosis. This infectious disease, which is often times shortened to TB, is commonly spread through the air and infects various parts of the body, mainly the lungs.The common symptoms of TB are: a bad cough that lasts more than three weeks, chest pains, coughing of blood, fatigue, weight loss, chills, fever, sweating at night, and loss of appetite. Most tuberculosis diagnosis are made through chest x-rays and microbiological cultures of bodily fluids. Tuberculosis is the second most common cause of death from infectious diseases. Due to lowered immune systems, it is highly present in areas of malnutrition and poverty along with those who have or at risk of having HIV. Many Asian and African countries are largely exposed to tuberculosis.Tuberculosis is not spread by physical contact like shaking someone’s hand, sharing food and drinks, kissing, etc. There are two types of tuberculosis related conditions: latent TB infection and TB disease. Latent TB infection is where an individual is not sick but is still infected withMycobacterium tuberculosis. The reason why the individual is not sick but still infected is due to their immune system which is preventing the growth ofMycobacterium tuberculosisand stops the spread of the bacteria to the environment. If theMycobacterium tuberculosisdoes grow, then the infection will proceed to the actual tuberculosis disease where the affected individual can now infect others. The reason for the bacteria growing may be a result of an HIV infection, recently infected with TB bacteria, alcohol or drug abuse, or other health problems. The treatment for latent TB infection consists of medicine called isoniazid (INH),rifampin (RIF), rifapentine (RPT). These medicines preventMycobacterium tuberculosisfrom growing and becoming active. The treatment for TB disease consists of an individual taking approximately seven drugs for 6 to 9 months. The main medicine that the individual must take include:isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA).
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) Essentiality of this protein: mixed essentiality
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Complex of proteins?: No, because chains A and B are both the same which means that it is a dimer. Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
0.5 *EC#: 6.3.2.4 Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=6.3.2.4
Figure 1: Reaction diagram of D-alanine:D-alanine ligase from BRENDA site.
Figure 2: PyMol images of D-alanine and ADP attached to Ddl. ADP is indicated by the enzyme with atoms colored in yellow; D-alanine is the enzyme shown in red. http://aac.asm.org/content/55/1/291.figures-only
-- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
Competitively inhibited by:
D-cycloserine - "preferential and weak inhibition at the second, lower-affinity binding site. D-cycloserine binding is tighter at higher ATP concentrations"
Expression Information (has it been expressed in bacterial cells):
Escherichia coli
Purification Method: Reading literature. Pending.
Image of protein (PyMol with features delineated and shown separately):
PyMol images of 3LWB (from left to right) shown in sticks with chains A and B shown in blue and green, and another image with the backbone in cartoon,
MSANDRRDRRVRVAVVFGGRSNEHAISCVSAGSILRNLDSRRFDVIAVGITPAGSWVLTD ANPDALTITNRELPQVKSGSGTELALPADPRRGGQLVSLPPGAGEVLESVDVVFPVLHGP YGEDGTIQGLLELAGVPYVGAGVLASAVGMDKEFTKKLLAADGLPVGAYAVLRPPRSTLH RQECERLGLPVFVKPARGGSSIGVSRVSSWDQLPAAVARARRHDPKVIVEAAISGRELEC GVLEMPDGTLEASTLGEIRVAGVRGREDSFYDFATKYLDDAAELDVPAKVDDQVAEAIRQ LAIRAFAAIDCRGLARVDFFLTDDGPVINEINTMPGFTTISMYPRMWAASGVDYPTLLAT MIETTLARGVGLH *length of your protein in Amino Acids = 373 *The beginning amino acid was switched from V to M.
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
39.710 kDa, or 39710.2 Da
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 27180
Abs 0.1% (=1 g/l) 0.684, assuming all pairs of Cys residues form cystines
Ext. coefficient 26930
Abs 0.1% (=1 g/l) 0.678, assuming all Cys residues are reduced
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
DNA sequence for Mycobacterium Tuberculosis from DNA Works Ouput. ATGTCTGCTAACGACCGCCGTGACCGTCGCGTTCGCGTCGCTGTTGTGTTCGGTGGCCGCTCTAACGAACACGCCATCTCTTGCGTTTCTGCGGGTTCTATCCTGCGTAATCTCGACTCT CGCCGTTTCGACGTTATCGCGGTAGGCATCACCCCGGCAGGCTCCTGGGTTCTGACCGATGCGAACCCGGACGCACTGACCATTACTAACCGTGAACTCCCGCAGGTAAAATCTGGTTCT GGTACGGAGCTGGCACTGCCTGCGGACCCTCGTCGTGGTGGCCAACTCGTTTCTCTCCCACCGGGTGCGGGCGAAGTTCTGGAATCTGTTGACGTTGTTTTCCCAGTTCTGCACGGCCCG TATGGTGAAGATGGTACGATCCAGGGCCTGCTGGAGCTCGCGGGTGTACCTTATGTTGGTGCAGGTGTTCTCGCGTCTGCGGTGGGTATGGACAAAGAATTCACCAAAAAACTGCTGGCA GCAGATGGCCTCCCAGTAGGTGCCTACGCGGTCCTCCGTCCGCCTCGTTCTACCCTCCATCGTCAAGAATGCGAGCGTCTGGGTCTGCCGGTTTTCGTTAAACCTGCCCGTGGCGGCTCT TCTATTGGTGTTTCTCGTGTTTCCTCTTGGGATCAGCTCCCTGCGGCAGTTGCGCGTGCCCGTCGCCATGACCCGAAAGTTATCGTTGAAGCGGCGATCTCTGGTCGTGAGCTGGAATGT GGTGTCCTGGAAATGCCGGACGGTACCCTCGAAGCGTCTACTCTGGGCGAAATTCGTGTGGCAGGCGTGCGTGGCCGTGAGGACTCTTTCTACGACTTTGCGACCAAATATCTGGACGAC GCAGCCGAACTGGATGTTCCGGCGAAGGTTGATGATCAGGTGGCTGAAGCTATCCGTCAGCTCGCAATTCGCGCATTCGCTGCGATTGACTGCCGTGGCCTCGCACGTGTGGACTTCTTC CTGACTGATGATGGTCCGGTGATCAACGAGATCAACACTATGCCGGGCTTTACTACCATTTCTATGTACCCGCGTATGTGGGCGGCATCTGGCGTTGATTACCCGACCCTGCTCGCGACC ATGATCGAAACCACCCTGGCTCGTGGCGTCGGTCTCCATTAA
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: Rv2981c (Tuberculist ID)
*Protein ID (NP or XP #) or Wolbachia#: N/A
*Organism (including strain): Mycobacterium tuberculosis
Etiologic Risk Group (see link below): 3
http://www.absa.org/riskgroups/bacteriasearch.php?genus=Mycobacterium&species=tuberculosis
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Mycobacterium tuberculosis is the leading bacteria that causes the pathogenic disease tuberculosis. This infectious disease, which is often times shortened to TB, is commonly spread through the air and infects various parts of the body, mainly the lungs.The common symptoms of TB are: a bad cough that lasts more than three weeks, chest pains, coughing of blood, fatigue, weight loss, chills, fever, sweating at night, and loss of appetite. Most tuberculosis diagnosis are made through chest x-rays and microbiological cultures of bodily fluids. Tuberculosis is the second most common cause of death from infectious diseases. Due to lowered immune systems, it is highly present in areas of malnutrition and poverty along with those who have or at risk of having HIV. Many Asian and African countries are largely exposed to tuberculosis.Tuberculosis is not spread by physical contact like shaking someone’s hand, sharing food and drinks, kissing, etc. There are two types of tuberculosis related conditions: latent TB infection and TB disease. Latent TB infection is where an individual is not sick but is still infected with Mycobacterium tuberculosis. The reason why the individual is not sick but still infected is due to their immune system which is preventing the growth of Mycobacterium tuberculosis and stops the spread of the bacteria to the environment. If the Mycobacterium tuberculosis does grow, then the infection will proceed to the actual tuberculosis disease where the affected individual can now infect others. The reason for the bacteria growing may be a result of an HIV infection, recently infected with TB bacteria, alcohol or drug abuse, or other health problems. The treatment for latent TB infection consists of medicine called isoniazid (INH),rifampin (RIF), rifapentine (RPT). These medicines prevent Mycobacterium tuberculosis from growing and becoming active. The treatment for TB disease consists of an individual taking approximately seven drugs for 6 to 9 months. The main medicine that the individual must take include:isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA).
Link to TDR Targets page (if present):
http://tdrtargets.org/targets/view?gene_id=5855
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein: mixed essentiality
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?: No, because chains A and B are both the same which means that it is a dimer.
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
0.5
*EC#: 6.3.2.4
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=6.3.2.4
Figure 1: Reaction diagram of D-alanine:D-alanine ligase from BRENDA site.
Figure 2: PyMol images of D-alanine and ADP attached to Ddl. ADP is indicated by the enzyme with atoms colored in yellow; D-alanine is the enzyme shown in red.
http://aac.asm.org/content/55/1/291.figures-only
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
http://aac.asm.org/content/55/1/291.long
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Use non-radioactive assay; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC149019/
malachite green assay
Structure (PDB or Homology model)
PDB #: 3LWB
Current Inhibitors:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=6.3.2.4
Competitively inhibited by:
D-cycloserine - "preferential and weak inhibition at the second, lower-affinity binding site. D-cycloserine binding is tighter at higher ATP concentrations"
Expression Information (has it been expressed in bacterial cells):
Escherichia coli
Purification Method:
Reading literature. Pending.
Image of protein (PyMol with features delineated and shown separately):
PyMol images of 3LWB (from left to right) shown in sticks with chains A and B shown in blue and green, and another image with the backbone in cartoon,
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
http://tuberculist.epfl.ch/quicksearch.php?gene+name=Rv2981c
MSANDRRDRRVRVAVVFGGRSNEHAISCVSAGSILRNLDSRRFDVIAVGITPAGSWVLTD
ANPDALTITNRELPQVKSGSGTELALPADPRRGGQLVSLPPGAGEVLESVDVVFPVLHGP
YGEDGTIQGLLELAGVPYVGAGVLASAVGMDKEFTKKLLAADGLPVGAYAVLRPPRSTLH
RQECERLGLPVFVKPARGGSSIGVSRVSSWDQLPAAVARARRHDPKVIVEAAISGRELEC
GVLEMPDGTLEASTLGEIRVAGVRGREDSFYDFATKYLDDAAELDVPAKVDDQVAEAIRQ
LAIRAFAAIDCRGLARVDFFLTDDGPVINEINTMPGFTTISMYPRMWAASGVDYPTLLAT
MIETTLARGVGLH
*length of your protein in Amino Acids = 373
*The beginning amino acid was switched from V to M.
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
39.710 kDa, or 39710.2 Da
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only): from Tuberculist
gtgagtgctaacgaccggcgtgatcggcgtgtccgcgttgccgtcgtgttcggcgggcgcagcaacgagcacgccatctcgtgtgtgtccgccggcagcatcctgcgcaacctggactcg
cggcggttcgacgtgatcgcggtgggtatcaccccggcaggttcgtgggtgctcaccgacgccaaccccgacgccctgacgatcaccaaccgggagcttcctcaggtcaaatcaggatcg
ggcaccgagctggcgctgccggccgatccgcggcgtggtggccagttggtgtcgctgccgcccggggccggcgaggttctggagtcggtcgacgtggtgttcccggtactgcacggcccgt
acggcgaggacggcacgatccagggactgctcgaactcgccggggtgccctacgtgggcgccggtgtgctggccagtgccgtcggcatggacaaggagttcaccaagaagctgctcgc
cgccgatggacttccggtgggtgcgtacgcggtgctgcgtccgccgcggtcgacactgcaccgccaggagtgcgaacggctgggcttaccggtgttcgtcaaacccgcccgaggcggctc
gtcgatcggtgttagccgggtgtcgagttgggatcaactgcccgccgcggtcgcgcgggcccgccggcatgaccctaaggtcatcgtcgaggccgcgatcagcggccgcgagctggaatgc
ggtgtgctcgaaatgccggacggcacactggaagccagcacgctgggggagatccgggtggccggggtgcggggacgcgaggactctttctacgacttcgcaaccaagtatctcgacgac
gcagccgaattggacgtgcccgccaaggtcgatgaccaggtcgcagaggcgattcgtcagctggcgatccgggcgttcgcggctatcgactgccggggtctggccagggtggacttcttcctc
accgacgacggtccggtgatcaacgagatcaacacgatgccgggattcaccacgatctcgatgtacccgcggatgtgggcggccagcggtgtcgactatccgaccctgctggcgacgatg
atcgagacgacattggcccgcggcgtgggcctgcactag
*GC% Content for gene: 67.47%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGTCTGCTAACGACCGCCGTGACCGTCGCGTTCGCGTCGCTGTTGTGTTCGGTGGCCGCTCTAACGAACACGCCATCTCTTGCGTTTCTGCGGGTTCTATCCTGCGTAATCTCGACTCT CGCCGTTTCGACGTTATCGCGGTAGGCATCACCCCGGCAGGCTCCTGGGTTCTGACCGATGCGAACCCGGACGCACTGACCATTACTAACCGTGAACTCCCGCAGGTAAAATCTGGTTCT GGTACGGAGCTGGCACTGCCTGCGGACCCTCGTCGTGGTGGCCAACTCGTTTCTCTCCCACCGGGTGCGGGCGAAGTTCTGGAATCTGTTGACGTTGTTTTCCCAGTTCTGCACGGCCCG TATGGTGAAGATGGTACGATCCAGGGCCTGCTGGAGCTCGCGGGTGTACCTTATGTTGGTGCAGGTGTTCTCGCGTCTGCGGTGGGTATGGACAAAGAATTCACCAAAAAACTGCTGGCA GCAGATGGCCTCCCAGTAGGTGCCTACGCGGTCCTCCGTCCGCCTCGTTCTACCCTCCATCGTCAAGAATGCGAGCGTCTGGGTCTGCCGGTTTTCGTTAAACCTGCCCGTGGCGGCTCT TCTATTGGTGTTTCTCGTGTTTCCTCTTGGGATCAGCTCCCTGCGGCAGTTGCGCGTGCCCGTCGCCATGACCCGAAAGTTATCGTTGAAGCGGCGATCTCTGGTCGTGAGCTGGAATGT GGTGTCCTGGAAATGCCGGACGGTACCCTCGAAGCGTCTACTCTGGGCGAAATTCGTGTGGCAGGCGTGCGTGGCCGTGAGGACTCTTTCTACGACTTTGCGACCAAATATCTGGACGAC GCAGCCGAACTGGATGTTCCGGCGAAGGTTGATGATCAGGTGGCTGAAGCTATCCGTCAGCTCGCAATTCGCGCATTCGCTGCGATTGACTGCCGTGGCCTCGCACGTGTGGACTTCTTC CTGACTGATGATGGTCCGGTGATCAACGAGATCAACACTATGCCGGGCTTTACTACCATTTCTATGTACCCGCGTATGTGGGCGGCATCTGGCGTTGATTACCCGACCCTGCTCGCGACC ATGATCGAAACCACCCTGGCTCGTGGCGTCGGTCTCCATTAA
*GC% Content for gene (codon optimized): 57.7%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
ATGTCTGCTAACGACCGCCGTGACCGTCGCGTTCGCGTCGCTGTTGTGTTCGGTGGCCGCTCTAACGAACACGCCATCTCTTGCGTTTCTGCGGGTTCTATCCTGCGTAATCTCGACTCT CGCCGTTTCGACGTTATCGCGGTAGGCATCACCCCGGCAGGCTCCTGGGTTCTGACCGATGCGAACCCGGACGCACTGACCATTACTAACCGTGAACTCCCGCAGGTAAAATCTGGTTCT GGTACGGAGCTGGCACTGCCTGCGGACCCTCGTCGTGGTGGCCAACTCGTTTCTCTCCCACCGGGTGCGGGCGAAGTTCTGGAATCTGTTGACGTTGTTTTCCCAGTTCTGCACGGCCCG TATGGTGAAGATGGTACGATCCAGGGCCTGCTGGAGCTCGCGGGTGTACCTTATGTTGGTGCAGGTGTTCTCGCGTCTGCGGTGGGTATGGACAAAGAATTCACCAAAAAACTGCTGGCA GCAGATGGCCTCCCAGTAGGTGCCTACGCGGTCCTCCGTCCGCCTCGTTCTACCCTCCATCGTCAAGAATGCGAGCGTCTGGGTCTGCCGGTTTTCGTTAAACCTGCCCGTGGCGGCTCT TCTATTGGTGTTTCTCGTGTTTCCTCTTGGGATCAGCTCCCTGCGGCAGTTGCGCGTGCCCGTCGCCATGACCCGAAAGTTATCGTTGAAGCGGCGATCTCTGGTCGTGAGCTGGAATGT GGTGTCCTGGAAATGCCGGACGGTACCCTCGAAGCGTCTACTCTGGGCGAAATTCGTGTGGCAGGCGTGCGTGGCCGTGAGGACTCTTTCTACGACTTTGCGACCAAATATCTGGACGAC GCAGCCGAACTGGATGTTCCGGCGAAGGTTGATGATCAGGTGGCTGAAGCTATCCGTCAGCTCGCAATTCGCGCATTCGCTGCGATTGACTGCCGTGGCCTCGCACGTGTGGACTTCTTC CTGACTGATGATGGTCCGGTGATCAACGAGATCAACACTATGCCGGGCTTTACTACCATTTCTATGTACCCGCGTATGTGGGCGGCATCTGGCGTTGATTACCCGACCCTGCTCGCGACC ATGATCGAAACCACCCTGGCTCGTGGCGTCGGTCTCCATTAA
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer:
TACTTCCAATCCATGTCTGCTAACGACCGCC
Reverse Complement Primer:
TATCCACCTTTACTGTTAATGGAGACCGACGCCAC
NCBI LINKS**
http://www.ncbi.nlm.nih.gov/gene/888415 - ddlA D-alanine--D-alanine ligase [ Mycobacterium tuberculosis H37Rv ]
http://www.ncbi.nlm.nih.gov/gene/13317778 - RVBD_2981c d-alanine ligase DdlA [ Mycobacterium tuberculosis H37Rv ]